Up To date, research for intracellular gene delivery has been conducted. Also, attempts to apply the research results to the development of practically available therapeutic agents have been conducted. Genes are genetic materials that contain nucleotides and polynucleotides, that is, nucleic acids. Genes has a strong negative charge because of the nucleotide unit. Genes are readily degraded by various enzymes that exist in vivo and in vitro, and have a short half life within cells or in vivo. Accordingly, all gene therapeutic agents based nucleic acid must be accompanied with development of therapeutic agents and delivery technology thereof.
Transfection is the process of introducing genetic agent into eukaryotic cells. It is the technology required in the research of basic sciences, including cytology, molecular biology, study of genetic functions and drug targets, etc. Also, it plays a core role for developing practical medicine in research of applied sciences including pharmacology, pharmaceutics, etc. Techniques for the delivery of genes into cells generally have been studied about the using of viral and non-viral carriers.
Currently, virus vector are used generally. The virus-mediated gene transfer has very high efficiency of gene transfer, but virus-mediated gene transfer may cause an immune response and thus is difficult to apply over a long period of time. In addition, it cannot completely exclude the possibility of pathogenic virus replication. In contrast, non-viral carriers have the advantage of delivering genetic materials without the risk of viral carriers (The AAPS Journal, 2010; 12: 492-503). In order to overcoming the risk of viral carriers, cationic lipids or polymers which are associable with anionic genes are used for non-viral delivery method.
U.S. Pat. No. 7,361,640 discloses claimed a non-covalent complex of gene/lipid/polycation, which is formed by adding a gene to a solution consisting of at least one the cationic lipid and the polycation. However, the cationic lipid species used in the complex exhibits cytotoxicity (Pharmaceutical Research 1994; 11: 1127-1131).
In addition, cationic lipids are not suitable for mass production because material need synthesis and a high degree of purification and is difficult to control their quality. In addition, it is difficult to commercialization because it is unstable during storage and distribution.
U.S. Patent Publication No. 2011/0038941 discloses the method for the preparing of oligonucleotide-lipid nanoparticle consisting of an oligonucleotide, a lipid and a complex agent, (1) mixing a lipid, a complexing agent and a cationic polymer in a water miscible organic solvent, (2) dissolving an oligonucleotide in an aqueous buffer, (3) injecting the mixture of step (1) into the mixture of step (2), or mixing the mixture of step (1) and the mixture of step (2) under pressure, to form a final mixture; and (4) removing the organic solvent from the final mixture to form the oligonucleotide-lipid nanoparticle. This preparing method is difficult to apply to industrial practice because it does not ensure the quality, it requires special instruments and mass production is difficult.
U.S. Patent Publication No. 2003/0068277 discloses the method for the preparing of complex composition of a polycationic complexing agent comprising protamine, spermine, spermidine, or chitosan and a phospholipid, but complex is designed for delivery of proteins to the pulmonary system by inhalation.
In preparing of gene delivery complex, chitosan is a cationic material which can be produced on a mass scale, abundantly found in nature, and is of high biocompatibility. Chitosan has attracted considerable interest as a non-viral carrier candidate for use in gene delivery. Notably, when administered to the body, chitosan is broken down by a lysozyme to amine sugar. It is a natural polymer which is as highly safe to the body as an LD50 of 16 g/kg in rats (Trends in Food Science & Technology, 2007; 18: 117-131).
Although it is considered as a potent candidate for a non-viral carrier which can form a complex with a gene through ionic bonds and transfer the gene into cells, chitosan is problematic in that its positive charge density is lowered at the physiological pH (pH 7˜7.4), which leads to poor binding affinity with genes. After all, chitosan does not maintain a complex form with genes in the physiological pH condition so that the genes dissociate from chitosan, with significantly poor delivery to cells.
Theerasak Rojanarata et al. prepared chitosan-thiamine pyrophosphate by using excess thiamine pyrophosphate as acid agent for dissolving chitosan, which is insoluble at the physiological pH and suggested the chitosan-thiamine pyrophosphate as a gene carrier (Pharmaceutical Research 2008; 25: 2807-2814).
The nanocomplex of chitosan and thiamine pyrophosphate, disclosed by Theerasak Rojanarata et al., was evaluated to deliver genes at high rates in vitro, but not in vivo. As opposed to in vitro, various serum proteins and cellular exudates exist in vivo. Therefore, when administered to the body, chitosan nanoparticles aggregate with serum proteins and cellular exudates in vivo and finally precipitate with the concomitant loss of gene delivery performance. For this reason, conventional chitosan complexes are reportedly known to exhibit poor gene delivery performance in vivo and to fail in commercialization as gene carriers (Expert Opinion on Drug Delivery, 2011; 8: 343-357).